The possibility of exposure to botulinum neurotoxin (BoNT), a powerful and potential bioterrorism agent, is considered to be ever increasing. The current gold-standard assay, live-mouse lethality, exhibits high sensitivity but has limitations including long assay times, whereas other assays evince rapidity but lack factors such as real-time monitoring or portability. In this study, we aimed to devise a novel detection system that could detect BoNT at below-nanomolar concentrations in the form of a stretchable biosensor. We used a field-effect transistor with a p-type channel and electrodes, along with a channel comprising aligned carbon nanotube layers to detect the type E light chain of BoNT (BoNT/E-Lc). The detection of BoNT/E-Lc entailed observing the cleavage of a unique peptide and the specific bonding between BoNT/E-Lc and antibody BoNT/E-Lc (Anti-BoNT/E-Lc). The unique peptide was cleaved by 60 pM BoNT/E-Lc; notably, 52 fM BoNT/E-Lc was detected within 1 min in the device with the antibody in the bent state. These results demonstrated that an all-carbon nanotube-based device  could be produced without a complicated fabrication process and could be used as a biosensor with high sensitivity, suggesting its potential development as a wearable BoNT biosensor. Clostridium bacteria, are considered the most poisonous protein products; these have 150 kDa molecular weight and seven structurally distinct serotypes (A to G) .,29.28,292 plasma treatment  was applied for 10 min. The CNT layer, to be used as the electrical leads, was attached to both ends of the high voltage/plasma-treated CNT channel. Then, 1 × 2 mm2 gold foil was attached by pressing onto these ends for contact with the probe measuring the electrical properties.After applying 50 V high voltage for 5 s using a high-voltage wideband amplifier , ON-hydroxysuccinimidyl pyrenebutanoate (Sigma-Aldrich) was used, and the prepared 6 mM succinimidyl ester solution was added dropwise into the CNT channel. After 3 h, the molecules that did not react were rinsed away with DMF. After washing the CNT surface with 10 mM HEPES buffer solution (pH 7.4), 0.8 μM peptide was incubated for 6 h. Determination of the concentration of the appropriate peptide for the experiment is shown in To demonstrate BoNT/E-Lc detection, the product peptides and Anti-BoNT/E-Lc were attached on the high voltage/plasma-treated CNT channel. First, to achieve immobilization of the peptides on the CNT surface, we used a non-covalent bonding method via the pyrene group of a succinimidyl ester ,30. To pTwo-probe electrical measurement was performed for testing the sensor’s electrical properties and protein sensing. Electrical measurements were performed by a voltage source , and a multimeter . Conductance vs. time data were recorded every 0.1 s with buffer and target protein flowing through the microfluidic channel. The reaction buffer solution flow began 1 min after the conductance vs. time recording started. The buffer solution was made to flow from the start of the experiment. When the sensor conductance stabilized, protein flowed in place of the buffer solution. All experiments were performed with a 0.01 μL/min flow rate using a syringe pump, using reaction buffer solutions suitable for each experiment; e.g., for observing peptide cleavage, 10 mM HEPES (pH 7.4) was used, whereas 50 mM Tris-buffer solution (pH 7.4) was used for observing specific bonding with the antibody. However, both solutions included 5 mM DTT and 10 mM Tween 20. To increase the activity of BoNT/E-Lc, BoNT/E-Lc samples were immersed in water with a constant temperature of 37 °C. Conductance vs. time recording was performed with the sensor placed on the heater to maintain a temperature of 37 °C. BoNT/E-Lc detection using antibody-antigen-specific binding was performed under room temperature, and the electrical signals were measured while attached to the vial surface (diameter 70 mm). A comparative experiment using BoNT/A-Lc was conducted using the same experimental conditions as the experiment targeting BoNT/E-Lc. The room temperature at which the experiment was performed was an average of 24 °C. Atomic force microscopy  was used for surface analysis of CNTs. X-ray photoelectron spectroscopy  was also performed to identify functional groups on the functionalized CNT surface.2 plasma to the CNT channel. Changes in the aligned CNT layers before and after high voltage and plasma treatment designed for CNT surface functionalization were confirmed using AFM and electrical characterization. As shown in 2 plasma treatment, indicating that the positive charges of the oxygen atoms had been transferred to the CNTs [2 plasma treatment using a back gate with SiO2 (120 nm) as a dielectric layer. For this experiment, a CNT-based FET device built on a Si/SiO2 substrate was employed.All-CNT-based devices for detecting BoNT/E-Lc were constructed using the production process shown in the CNTs ,33. We mD is the drain current, VG is the gate voltage, L is the channel length, W denotes a channel width, and VD denotes a drain voltage [2, ε0 is the vacuum permittivity, and t is the thickness of the gate dielectric. The effective device mobility estimated based on these equations was ≈ 1.3 × 104 cm2V−1s−1 .Based on the measurement results, we next attempted to obtain the effective device mobility  180 and isoleucine (I) 181 in the SNAP-25 sequence ,22,35. UThe electrical response of the sensor at six different BoNT/E-Lc concentrations  is shown in The times required from injection of BoNT/E-Lc to the saturation of conductivity were 3 min at 3 nM and 38.3 min at 60 pM b,f. HoweFinally, we confirmed the selectivity of BoNT/E-Lc for the unique peptide. 3 nM BoNT/A-Lc was selected as the target substance, as it can cleave SNAP-25 similarly to BoNT/E-LC . As showIn conclusion, specific reactions between the product peptides and BoNT/E-Lc were demonstrated, confirming the utility of the devised system for this purpose.We also conducted experiments for BoNT/E-Lc detection using antigen-antibody interaction. This method can be performed at room temperature and is adapted in many biosensors owing to its relatively straightforward mechanism, which constitutes specific binding between the epitopes found on the antigen and the binding site on the antibody. This experiment was initiated by attaching the PDMS cover with a microfluidic channel onto the CNT-based device with the Anti-BoNT/E-Lc. With the manufactured microfluidic device in the bent state, the conductivity change was checked in real time with 500, 300, 100, 70 and 52 fM BoNT/E-Lc a–c. When50) of BoNT. In particular, we also showed that detection of biomolecules through the microfluidic channel is possible even in the bent state.BoNT/A-Lc was also used to determine the selectivity of CNT-based sensors. Upon 500 fM BoNT/A-Lc injection, no changes in electrical signal were observed e. AlthouThe XPS was used to investigate the interaction of the molecules that occurred on the surface of the CNTs. In this experiment, the main task of XPS is the observation of nitrogen introduced by biomolecules. The binding energy of nitrogen is ~400.4 eV .We produced a new type of all-CNT-based device using a simple production process. Using this device, we attempted to detect BoNT/E, which is emerging as one of the candidates of biochemical warfare. By monitoring the cleavage of a unique peptide and the specific binding between BoNT/E and Anti-BoNT, we could confirm the high sensitivity of the all-CNT device. Through multiple BoNT/E detection experiments, we showed the potential for expanding the biosensor industry as well as the possibility of producing a biosensor that can detect biomaterials within a short time to counteract possible biological weapons. Although developing a device with consistently high performance is beset by difficulties, our results may provide the basis for the fabrication of a CNT-based device that will not require an alignment process of CNTs or the transfer of grown CNTs. Furthermore, we suggest that it may also enable the fabrication of a wearable biosensor.


P = 0.002). Multivariate survival analysis indicated that LINC00957 was a prognostic factor for CRC patients (P = 0.010). Mechanically, inhibition of LINC00957 expression reversed 5-FU resistance by down-regulating P-gP. In summary, our study indicated that this novel lncRNA expression signature might be a useful biomarker of the prognosis and therapeutic target for CRC patients.Colorectal cancer (CRC) is one of the most prevalent digestive tumors in China. Recent studies indicate that long intergenic non-coding RNAs (lincRNAs) play a crucial role in predicting survival for CRC patients. However, the novel lincRNA, LINC00957, is largely unclear in CRC. The purpose of the current study was to determine LINC00957 expression, assess its the clinical significance and explore the potential mechanism in CRC. The qRT-PCR was used to quantify the expression levels of LINC00957 in tissues and cell lines. Our research revealed that LINC00957 was significantly higher expression in CRC. In addition, the LINC00957 expression was associated with TNM stage and chemotherapy outcome, but age, gender, tumor size, histological grade, primary tumor location. CRC patients with high LINC00957 expression level showed poor overall survival ( Colorectal cancer (CRC) has been reported to be one of the most common malignant tumors in the digestive system, with a high recurrence and cancer-related mortality rate worldwide . In ChinIn this study, we explore its function in CRC. Firstly, we analyzed lincRNA expression profiles in 440 CRC patients from The Cancer Genome Atlas (TCGA) and identified it was significantly associated with survival. Then we further examined the expression of LINC00957 in CRC tissues and cell lines compared to their matched adjacent non-tumor by qRT-PCR. Besides, we also analyzed the association between the LINC00957 expression and the clinic pathological characteristics of CRC patients. Mechanically, we explored that its function in 5-FU resistance was associated with multi-drug resistance relative genes. All data demonstrated that it was up-regulated in CRC, and was associated with TNM stage, chemotherapy outcome, but not with the age, gender, tumor size, histological grade, and primary tumor location. What's more, the high expression of LINC00957 suggested poor survival in CRC and involved in drug-resistance by regulating P-gp expression. Taken together, our study suggested that the upregulation of LINC00957 played a promoting role in CRC and it would be a potential biomarker for CRC progress and therapeutic target of CRC.one hundred and sixty pairs of CRC tissues and adjacent normal tissues involved in present study were collected from Affiliated Hospital of Jiangnan University. Informed consents to approve our use of these tissues in the research were obtained from all patients. Patients who postoperatively received 5-Fu-based first-line systematic chemotherapy were enrolled in the present study. The protocol was approved by Affiliated Hospital of Jiangnan University. All methods involving human patients were performed in accordance with the relevant guidelines and regulations of Affiliated Hospital of Jiangnan University. These specimens were confirmed by histopathological examination and immediately stored at −80°C until use.Tumor assessment was performed after every 2 cycles of chemotherapy according to the Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1) criteria (22), and the assessment was classified as a complete response (CR), a partial response (PR), stable disease (SD) and progressive disease (PD).2 in a humidified atmosphere.Human CRC cell lines  and normal human colon epithelial cells NCM460 were purchased from Nanjing Musai Bio-Tech Co, Ltd . Fluorouracil (5-Fu)-resistant HCT-8 cells (HCT-8/5-Fu) (KG333) was purchased from Keygen Biotech Co. Ltd. 5-Fu-resistant LoVo cells (LoVo/5-Fu) were derived by treating LoVo cells with stepwise increasing concentrations of 5-Fu  over 6 months. 5-Fu-resistant CRC cells were cultured as we reported previously . All celCt−Δ method was used to calculate expression levels and 2Ct−ΔΔ method was used to calculate relative expression level. GAPDH was used as an internal control , according to the manufacture's protocol. The concentration and purity of total RNA were measured on a nanodrop spectrophotometer . For qRT-PCR, RNA was reverse transcribed to cDNA by using a PrimeScript RT reagent Kit . qRT-PCR was performed using a SYBR_Premix ExTaqII kit  in the CFX96 Real-Time PCR Detection System  to determine the relative expression levels of target genes. The reaction conditions was 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. The 2 control . The seqLOVO/5-FU and HCT8/5-Fu cells on 50–70% confluence were treated with LINC00957-siRNA1 , LINC00957-siRNA2 , LINC00957-siRNA3  and nontargeting siRNA (si-NC), respectively. Cells were transfected with siRNA by using Lipofectamine 4000  according to the manufacturer's instructions. The cells were harvested for further assays 48 h after transfection. Expression of LINC00957 were determined by Real-time PCR. Three independent experiments were performed, and the data are expressed as the mean ± standard error of the mean (SEM).5cells/200 ul) were seeded in 96-well plates and placed in an incubator at 37°C for 12 h. The medium was then replaced with medium containing different concentrations of 5-FU. After 48 h, the medium of each well was added with 10 uL CTB. After incubation for 4 h, the plates were read on a microplate reader. Fluorescence density at 560 nm excitation and 590 nm emission was determined for statistical analysis. Three independent experiments were performed, and the data are expressed as the mean ± standard error of the mean (SEM).These cytotoxic effects of 5-FU were evaluated at 48 h with the CTB  assay. Cells  and anti-GAPDH were used to detect the expression of the proteins. The antigen-antibody complexes were visualized by an enhanced chemiluminescent reaction. Protein bands were analyzed using Image J software . Three independent experiments were performed, and the data are expressed as the mean ± standard error of the mean (SEM).https://gtexportal.org/).The LINC00957 gene expression in different tissues was from The Genotype-Tissue Expression (GTEx) project  was been used. Then all data for individual cancers from TCGA were downloaded.Data in OncoLnc from the website (http://tcga-data.nci.nih.gov./tcga/). The STAD-associated gene clusters and pathways were identified in the c2.cp.kegg.v5.1.symbols.gmt data set from the Msig database using GSEA version 3.0. Enrichment analysis was performed with random combination number of 1,000 and false discovery rate (FDR) <0.05 as the criteria for significantly enriched genes. The gene sets were classified into the LINC00957 high- and low-expression groups based on its median expression level, and the effect of LINC00957 expression was analyzed.The RNA-sequencing V2 datasets and clinical data of STAD samples were downloaded using the Bioconductor/TCGA Biolinks function package from TCGA database  was calculated using the Kaplan-Meier method, and the results of the analysis were considered significant in a log-rank test if P < 0.05.The expression levels of LINC00957 in CRC tissues were analyzed by unpaired P > 0.05, P < 0.05, In order to explore LINC00957 expression, GTEx database was used to present the gene expression in different human tissues. It was showed that LINC00957 was abnormal expression in different tissues as P < 0.001). We further examined its expression in the normal human colon epithelial cell line NCM460 and CRC cell lines . As shown in In this study, we firstly compared the expression of LINC00957 by qRT-PCR in 160 cases of CRC tissues and their matched adjacent non-tumor tissues. As shown in P = 0.040) and chemotherapy outcome (P = 0.026). However, the level of LINC00957 expression in CRC tissues was not associated with the age, gender, tumor size, histological grade, primary tumor location . At the same time, we analyzed the relationship of LINC00957 expression levels and prognosis in CRC patients from TCGA database. LINC00957 expression showed shorter survival time than those with low LINC00957 expression in P = 0.03). We then analyzed the factors of poor prognosis by univariate and multivariate cox proportional hazards analyses. In univariate overall survival (OS) analysis, we observed that LINC00957 expression (P < 0.001), TNM stage (P < 0.001), chemotherapy outcome (P = 0.014) and gender (P = 0.048) retained significance as prognostic factors (P = 0.010) and chemotherapy outcome (P = 0.018) were an independent predictor of poor prognosis. Accordingly, our data demonstrated that high expression of LINC00957 could predicate a poor prognosis of patients with CRC.The relationship between the LINC00957 expression and the survival time of CRC patients was further analyzed. Our data showed that CRC patients with high LINC00957 expression appeared a worse prognosis when compared with those with low level in  factors . In multP < 0.001). According to the CTB experiment, the IC50 of LOVO was 428.5 ng/mL (LogIC50 = 2.632 ± 0.051); the IC50 of LOVO/5-FU was 1621 ng/mL (LogIC50 = 3.210 ± 0.048); the IC50 of LOVO/5-FU/si-RNA2 was 590.1 ng/mL (LogIC50 = 2.771 ± 0.061). The IC50 of HCT was 409.8 ng/mL (LogIC50 = 2.613 ± 0.052); the IC50 of HCT/5-FU was 3064 ng/mL (LogIC50 = 3.486 ± 0.045); the IC50 of HCT/5-FU/si-RNA2 was 941.3 ng/mL (LogIC50 = 2.974 ± 0.096). As shown in P < 0.05, The two 5-Fluorouracil-resistant model cell lines (LOVO/5-FU and HCT8/5-FU) were used to study the resistance mechanism of LINC00957. Firstly, three pairs of silence primers of LINC00957 were designed to reduce the expression of LINC00957 in both resistant cells. As showed in in vitro and in vivo  expression as a novel prognostic biomarker in colorectal cancer (CRC). Aberrant expression of LINC00957 was associated with CRC in GTEx dataset, and his upregulation was confirmed by qRT-PCR in patients' tumor and paired normal tissues. Expression of LINC00957 was significantly associated with advanced TNM stage, poor chemotherapy outcomes, and worse prognosis. Thus, we speculated that LINC00957 was involved in tumor progression and development of multidrug resistance. The latter hypothesis was partially demonstrated using two 5-Fluorouracil-resistant model cell lines (LOVO/5-FU and HCT8/5-FU), in which the silencing of LINC00957 was able to reverse 5-FU resistance, potentially through P-gP modulation. Beyond the functional study using knockdown experiments, this study also shows the potential role of the linc00975 in drug resistance. All these data together suggest a role of LINC00957 as oncogenic gene in CRC. Overall, this study is novel and well-performed, representing a good starting point for the exploration of the role of LINC00975 in CRC. The results obtained from CRC patients' analysis are a reasonable base for further experiments.This research had some limitations. Firstly, the study is very linear, clear and simple. However, the evidences supporting the involvement of LINC00975 overexpression in the development of a drug-resistance phenotype are poor. Additional and more pround experiments are needed to sustain with mechanistic evidences, the designation of LINC00975 expression as a potential therapeutic target for CRC. Secondly, all experiments have been performed well and reasonably. Although the functional mechanism of linc00975 in CRC development and drug-resistance have not been investigated, it could be continued in the further studies.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.DH and LZ designed the study. LZ and LL developed the methodology, performed the analysis, and wrote the manuscript. PZ, YC, and LL collected the data, cultured cell lines and did PCR, CTB, Western blot assays.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Two readers independently assessed subjective image quality using a five-point Likert scale . Inter-observer agreement was calculated and expressed as percentage of agreement. Noise was determined for calculation of signal-to-noise-ratio (SNR). Effective radiation dose (ED) was calculated from the dose-length-product (DLP).All examinations showed diagnostic image quality regarding evaluation of inflammatory sinus disease. On average, subjective general image quality was rated moderate (= 3) with a percentage of agreement between the observers of 81%. The mean image noise was 14.3 HU. The calculated median SNR was 6.0 for intraorbital fat, and 3.6 for the vitreous body, respectively. The median DLP was 2.1 mGy*cm, resulting in a median ED of 0.012 mSv.Taking the study limitations into account, ultra-low-dose tin-filtered CT of the paranasal sinus at a tube voltage of 100 kV utilizing an iterative reconstruction algorithm provides for reliable exclusion of suspected acute inflammatory sinus disease in 100% of the cases. The tin-filter absorbs low-energy photons that are less relevant in a high-contrast setting (e.g. bone imaging) and, therefore, helps to reduce the radiation exposure of the patient. Along with a tube-voltage of 100 kV, reduced tube-current and the use of iterative reconstruction this setting poses a promising new perspective in low-dose imaging of the paranasal sinus. The positive effects of tin-filtration on image quality and radiation dose in parasinus CT have been shown in cadaver studies ) was measured by one reader using a circular region-of-interest (ROI). To account for anatomic differences between patients the ROI was chosen as large as possible, while carefully avoiding adjacent structures and image artifact in order to prevent partial volume effect. Noise (N) was determined as the standard deviation of air measured anteriorly to the face on the level of the maxillary sinus using a 20 mm ROI. The measurement was repeated twice (ROI placed in front of the right and left maxillary sinus), and the mean result was used for further calculations. Based on these measurements, signal-to-noise ratio (SNR) was calculated as following: SNR = attenuation / [(N of air measured anteriorly to the right face + N of air measured anteriorly of the left face) x 0.5].-1 x cm-1; referenced to the 16-cm CTDI-phantom) was used for estimation of the ED . The resulting median effective radiation dose was 0.012  mSv [range 0.010–0.022 mSv].The percentage of agreement for general image quality was moderate (81% agreement) with moderate reliability . Details concerning the percentage of agreement of the individual anatomical structures are given in The general image quality was rated 3 by both readers . DetailsThe mean noise was 14.3 ± 2.5 HU [range 9.4–24.2 HU]. The attenuation of the intraorbital fat was -85.3 ± 9.0 HU [range -66.0 –-111.0 HU] and 51.4 ± 9.3 HU [range 33.0–74.0] for the vitreous body. The calculated median SNR for intraorbital fat was 6.0  [range 3.4–9.4], and 3.6  [range 2.1–6.2] for the vitreous body, respectively.The eye lenses as radiosensitive organs are exposed to direct and scattered radiation during CT of the paranasal sinus ,11,13,14Previous studies demonstrated promising results with different dose reduction strategies, including high pitch scanning ,6, low-klow-dose” CT relating to the paranasal sinuses in 1991, they specified that 23 mAs at a standard tube-voltage of 120 kV provides sufficient quality for the evaluation of suspected sinusitis [nd generation dual-source CT-scanner [ultra-low-dose” CT [low” has changed during the past 2 decades. For a more tangible comparison, the annual natural radiation exposure in Germany is 2.1 mSv , which is approximately half of the ED dose of our present protocol [When Marmolya et al. first introduced the term “inusitis . Bulla e-scanner . In 2012dose” CT  which wadose” CT . A mean dose” CT . With a dose” CT ,14. Howeprotocol . Althougvs. air, mucosal swelling or fluid–image quality is primarily limited by the image noise and not the image contrast.Moreover, there are additional benefits of keeping the kV high (e.g. 100 kV) and simultaneously reducing the X-ray flux with additional tin-filtration, compared to the more conventional approach of lowering the kV to reduce the X-ray flux. Higher photon energies can decrease image noise measured in the detector because of reduced absorption in the patient. This issue is especially important in paranasal CT imaging, as due to the high intrinsic contrast between relevant structures–bone Beside detection or rule out of acute inflammatory sinus disease, the present ultra-low-dose protocol might be of special interest when children are assigned for CT imaging of the paranasal sinus. Compared with the European reference dose for CT examination of the face and sinus (DLP 360 mGycm), the 100 kV tin-filter technique with 35 mAs cuts the radiation exposure of the patient down to about 0.6% in the present study .The present study has several limitations. First, a tin-filter for x-ray prefiltration is not available on the vast majority of CT scanners at the moment. Second, this study only demonstrates the feasibility of a tin-filtered protocol at a specified tube-voltage of 100 kV. The potential for further optimization of image quality and / or radiation dose by tin-filtration at different tube voltages remains unclear and should be evaluated in further studies, which then should also include control-groups. Third, due to the combination of different dose saving strategies (tin-filter / low kV / IR) we cannot declare which of the individual investigations is mainly responsible for the achievement in dose reduction. Fourth, image quality of our proposed protocol was considered to allow a sufficient diagnosis of inflammatory sinus disease. For detailed depiction of anatomic information prior to surgery, in trauma settings, or for planning of 3D surgical navigation the current protocol was not evaluated and might not provide the image quality necessary for these indications. Because of the fact that only one group was evaluated in the current study, the visual score was not based on relatively high-dose CT or standard low-dose CT, respectively. Therefore, theoretically there was a possibility of misdiagnosis in the current study. Last, we used a more up-to-date conversion factor of 0.0019 mSv/mGy*cm for estimation of the effective radiation dose as proposed by Deak et al. in 2010 , while tTaking the study limitations into account, ultra-low-dose tin-filtered CT of the paranasal sinus at a tube voltage of 100 kV utilizing an iterative reconstruction algorithm provides for reliable exclusion of suspected acute inflammatory sinus disease in 100% of the cases at a mean effective dose of 0.012 mSv per scan.


The findings of the study highlight the capability of artificial gravity (2-g at feet) created via SAHC toward evoking blood pressure regulatory controls analogous to standing, therefore, a potential utility toward mitigating deleterious effects of microgravity on cardiovascular performance and minimizing post-flight orthostatic intolerance in astronauts.Autonomic control of blood pressure is essential toward maintenance of cerebral perfusion during standing, failure of which could lead to fainting. Long-term exposure to microgravity deteriorates autonomic control of blood pressure. Consequently, astronauts experience orthostatic intolerance on their return to gravitational environment. Ground-based studies suggest sporadic training in artificial hypergravity can mitigate spaceflight deconditioning. In this regard, short-arm human centrifuge (SAHC), capable of creating artificial hypergravity of different g-loads, provides an auspicious training tool. Here, we compare autonomic control of blood pressure during centrifugation creating 1-g and 2-g at feet with standing in natural gravity. Continuous blood pressure was acquired simultaneously from 13 healthy participants during supine baseline, standing, supine recovery, centrifugation of 1-g, and 2-g, from which heart rate (RR) and systolic blood pressure (SBP) were derived. The autonomic blood pressure regulation was assessed via spectral analysis of RR and SBP, spontaneous baroreflex sensitivity, and non-linear heart rate and blood pressure causality (RR↔SBP). While majority of these blood pressure regulatory indices were significantly different ( CentralTherefore, to cope with the effects of orthostatic challenge on physiological performance, autonomic, baroreceptor, and vasomotor controls play a consequential role to various degrees. Enfeebled or impaired blood pressure regulatory controls owing to long-term microgravity exposure could lead to an abrupt decline in arterial blood pressure on assuming upright stance resulting in reduced cerebral perfusion . Thus, iGround-based experiments, performed to comprehend physiological response to microgravity simulated via bed rest immobilization  and poteIn the previous work from our group , we demoThe detailed experimental protocol has been explained in the previous work from our group . Here, wIn the centrifuge, the participant was strapped with their head near the center of 2.8-m radius centrifuge and feet outwards. The participant remained supine (Baseline) in the centrifuge for 20 min of baseline recording. After completion of baseline, the participant was transitioned with assistance into the standing position for 5 min (stand test). After the stand test, the participant lay supine (recovery) in the centrifuge for another 15 min, after which the centrifuge was ramped up to a rate that applied 1-g at feet  for 5 min and then increased to 2-g at feet  for 5 min. Following 5 min of centrifugation at 2-g, the centrifuge rotation was slowed and halted in 30 s.Table 1. Continuous blood pressure was acquired from non-invasive finger photoplethysmography cuff  using NI data acquisition  system at a sampling rate of 1000 Hz. Ethics approval for experimentation was obtained from the University of Toulouse. Experimentation complied with rules and regulations set forth by the research ethics board of the University of Toulouse. Written and informed consent form for participation was obtained from each participant prior to any experimentation.Data was acquired from 13 participants . The detailed demographic information is provided in Short-arm human centrifuge is a training tool capable of creating artificial gravity of different g-load . Brieflyleration . The cenDiastolic nadirs of each beat were first detected from the continuous blood pressure waveform to obtain diastolic blood pressure (DBP). The R-R time interval was obtained as the duration between two adjacent diastolic nadirs. Systolic blood pressure (SBP) was obtained as the maximum blood pressure between two adjacent diastolic nadirs. Beat-by-beat mean arterial pressure (MAP) was derived from blood pressure waveform as a mean value between two adjacent DBP locations. 5-min of data from each experimental condition was considered for analysis.nu = PV LF÷Total Power, LFnu = PLF÷Total Power, and HFnu = PHF÷Total Power, in case of RR signal the normalized power in only LF and HF frequency band was calculated as LFnu = PLF÷ and HFnu = PHF÷ where Total Power = PV LF+PLF+PHF in accordance with recommendation in the literature  of RR and SBP was calculated in very low frequency , low frequency , and high frequency  bands. There upon, the SBP power distributed (P) in the respective bands were normalized as VLFterature . The PSD1 similar to other research in the literature , signifying the feedforward (non-baroreflex) and feedback (baroreflex) controls of blood pressure was obtained using convergent cross mapping (CCM) similar to our previous work . Prior tpost hoc analysis using Tukey-HSD method to account for the significance of changes in the cardiovascular parameters and blood pressure regulatory indices during different experimental conditions. The test of significance was conducted using a statistical toolbox of MATLAB . The test results at α = 0.05 were considered significant. All tabular data in the article are presented as mean ± SD unless mentioned otherwise.Test for normal distribution of the mean of different variables studied in this research was conducted using Shapiro–Wilk test . A multiple comparison test was conducted using repeated measure of ANOVA  or Friedman test  followed by post hoc analysis using Tukey-HSD method was conducted to account for difference exerted by different experimental conditions on the cardiovascular parameters as well as on the blood pressure regulatory indices. Table 2 summarizes the behavior of cardiovascular parameters during baseline, standing, recovery, centrifugation of 1-g, and 2-g. Stand test or application of centrifugation inflicted no change in SBP (p = 0.13), DBP (p = 0.27), or MAP (p = 0.28). RR intervals reduced significantly during standing (p < 0.001) and 2-g (p < 0.001) compared to supine. Additionally, RR intervals reduced significantly during standing (p < 0.001) and 2-g (p < 0.001) compared to recovery as well as during 2-g (p = 0.02) compared to 1-g. No change in RR intervals was obtained during recovery (p = 0.99) and 1-g (p = 0.09) compared to baseline. Furthermore, no change (p = 0.57) in RR intervals was observed between standing and 2-g. Figure 1 shows an example of RR intervals and blood pressure dynamics in response to different experimental conditions.Test of normality resulted in data exhibiting mixed behavior given the limited sample size, therefore, Friedman test followed by Figure 2 summarizes the normalized spectral power distribution in the VLF, LF, and HF bands of SBP and LF, HF, and LF/HF ratio of RR intervals. The spectral power distribution in the respective frequency bands of SBP and RR in absolute and normalized units is summarized in Table 3. Table 4 lists the post hoc comparison p-value between experimental conditions for respective frequency bands. Significant change was observed in low-frequency SBP power both in normalized and absolute units during standing (p = 0.04 and p = 0.01) and 2-g (p < 0.001 and p < 0.001) compared to baseline. Moreover, the low-frequency SBP power  was significantly different during standing and 2-g compared to recovery. In addition, the SBPLF and SBPHF in absolute power were observed to be significantly different (p < 0.05) at 2-g compared to 1-g (Table 3). No difference (p > 0.10) was the behavior of such variable at 2-g compared to standing (Table 4).p = 0.007) and decreased (p = 0.007) significantly only at 2-g compared to baseline (Table 4). The LF/HF ratio increased significantly at 2-g (p = 0.007) compared to baseline. Moreover, the three metrics were significantly different (p < 0.05) during standing and 2-g compared to recovery (Table 3). In terms of absolute power no change (p = 0.87) was observed in the RRLF, while RRHF decreased significantly (p < 0.05) during standing and 2-g compared to baseline and recovery (Table 3). No difference (p > 0.10) in the dynamics of RR spectral power was obtained at 2-g compared to standing (Table 4).The spectral power (n.u.) in the LF and HF frequency bands of RR intervals increased  and 2-g  compared to baseline. Significant decline in BRS was observed during standing  and 2-g  compared to recovery as well as at 2-g  compared to 1-g (Table 3). No change  in BRS was observed at 2-g compared to standing (Table 4). The distribution of up slope and down slope BRS for the study participants is detailed in Figure 3.Baroreflex sensitivity decreased (both up and down slope) significantly during standing  and 1-g centrifugation (p = 0.04), a significant increase in baroreflex arm of the interaction compared to supine was observed, while no change (p = 0.26) in the non-baroreflex arm of the interaction was observed during experimental conditions. No change (p = 0.16) was observed in the baroreflex arm of the interaction at 2-g compared to baseline. Additionally, no difference between the standing and 2-g centrifugation was observed in the baroreflex arm .At supine rest, the non-baroreflex arm of the heart rate and blood pressure interaction was significantly higher than the baroreflex arm. During standing (The current research investigated the capability of centrifugation (1-g and 2-g) at feet to evoke autonomic control of blood pressure analogous to standing. The autonomic blood pressure regulation mechanisms via causal heart rate-blood pressure interaction, spontaneous baroreflex sensitivity, and spectral analysis of SBP and RR were studied. The analysis results ascertained previously contemplated yet undocumented potential of SAHC to evoke autonomic blood pressure control analogous to standing in natural gravity, therefore, a potential utility toward minimizing the adverse effects of long-term microgravity exposure on the cardiovascular performance, hence, minimizing orthostatic intolerance in astronauts upon return to Earth.The success of future inter-planetary missions to Mars depends on the design of pertinent countermeasures to mitigate the adverse effects of spaceflight deconditioning. The shift of central blood volume above thoracic leading to increased ventricular filling, stroke volume, and cerebral blood flow is an immediate consequence of physiological adaptation to microgravity. Accordingly, baroreceptor unloading, autonomic sympathetic nerve activity, and the vasomotor control remain vastly inhibited for the duration of spaceflight . The proFurthermore, decreased blood flow to the peripheral regions, especially to the calf skeletal muscles, render posture muscle group with nutritional scarcity, and is a major contributor toward skeletal muscle atrophy, bone remodeling, and decline in the calf circumference . While aThus, an external system capable of evoking autonomic control of blood pressure  and simultaneously increasing blood flow to the calf musculature to assure adequate nutritional and metabolic supply can mitigate the deterioration of cardiovascular performance associated with long-term exposure to microgravity.Exercise training and lower-body negative pressurization have been utilized as a potential countermeasure to spaceflight deconditioning. However, traditionally used aerobic exercise, resistance training, and lower-body suction has been limited in effect due to their inability to challenge multiple physiological systems that are associated with standing . ConsequTables 4. The results suggest the dynamics of studied variables were not different between the two conditions, therefore, the residual effects of standing was minimized and not likely to influence the autonomic behavior during centrifugation. The experimental conditions inflicted no change in SBP (p = 0.13), DBP (p = 0.27), or MAP (p = 0.28). This observation suggests that blood pressure was well-regulated during orthostatic challenge evoked via standing and centrifugation by active autonomic control of blood pressure. A significant decline was observed in the power distributed in the HF band of RR during standing (p = 0.02) and 2-g (p = 0.001) compared to baseline (Table 4), suggesting withdrawal of vagal nerve activity; resulting in a shift of sympatho-vagal balance toward sympathetic activity. Also, increase in LF SBP power was observed during standing and 2-g compared to supine (Table 3). Additionally, baroreflex sensitivity (both up slope and down slope) declined during standing and 2-g compared to supine (Table 3), which is the result of decreased vagal activity (Table 4) and increased heart rate or reduced RR intervals (Table 1) contributing toward maintenance of blood pressure equilibrium. While these markers of autonomic controls of blood pressure were significantly different from baseline during standing and 2-g, no change (p > 0.10) in the autonomic blood pressure control between standing and 2-g was observed (Table 4). Moreover, no change (Table 4) was observed in these indices between baseline and 1-g. This observation suggests 2-g is more closely related to standing compared to 1-g in terms of stimulating autonomic blood pressure regulatory controls.We applied external perturbation to the hemodynamic homeostasis via stand test and centrifugation of 1-g and 2-g at feet. A recovery period followed a stand test to minimize its influence on the autonomic behavior during centrifugation. The comparison result of baseline and recovery period is summarized in While the spectral analysis of SBP and RR time series and baroreflex sensitivity are well-accepted norm to account for the autonomic control of blood pressure, criticism of such approaches in the literature is also prevalent for the inability of the spectral method to account for the non-linearity of underlying physiology and BRS for not able to address the closed loop heart rate and blood pressure interaction . As suchFigure 4. No change (p > 0.05) was observed in the dynamics of non-baroreflex (RR→SBP) causality during standing or 2-g compared to supine. However, a significant increase was observed in the baroreflex (SBP→RR) causality; both during standing (p = 0.009) and 1-g (p = 0.04), Table 4. No change (p = 0.16) was observed in SBP→RR causality during 2-g compared to baseline. The slight decline in baroreflex causality at 2-g compared to 1-g (Table 3) could be due to the fact that 2-g was more stressful than standing and 1-g evident from certain study variables , which changed only at 2-g compared to baseline. Furthermore, heart rate, BRS, SBPLF, and SBPHF changed significantly at 2-g compared to 1-g (Table 3), which further hints toward 2-g being more stressful than 1-g. The RR intervals reduced at 1-g (p = 0.09) albeit not significantly but given limited sample size it does hint toward 1-g being more challenging than baseline. The baroreflex coupling is observed to decline before syncope (p = 0.80) or baroreflex (p = 0.88) causal events during centrifugation compared to standing (Table 3). Therefore, the observations of current study suggest centrifugation at feet is capable of evoking autonomic control of blood pressure analogous to standing.The results of closed loop heart rate and blood pressure interaction are detailed in  syncope , in our Table 4), which indicate 2-g was more stressful than standing. Accurate information regarding the degree of blood pooling achieved during each experimental condition will shed further light pertaining to the vigor of 2-g in relation to standing.Experimental protocol in the current study was not randomized, i.e., centrifugation always followed the stand test. Although a 15-min recovery period was adopted to minimize the effects of stand test on centrifugation, in future study randomized experimental protocol should be considered. Furthermore, due to the unavailability of the respiration signal, the role of hypergravity toward the dynamics of respiration could not be studied. Respiration is known to affect both heart rate (RR) as well as blood pressure . AdditioMoreover, additional mechanisms that account for blood pressure regulation such as skeletal muscle pump  shall also be investigated and compared in the future . FurtherCardiovascular adaptation to microgravity impairs autonomic control of blood pressure, consequently, astronauts are susceptible to orthostatic intolerance on return to gravitational environment. Sporadic training in artificial hypergravity is proposed to mitigate the effects of spaceflight deconditioning. SAHC is a promising tool for simulating artificial gravity of different g-loads. The response of blood pressure regulatory controls to simulated hypergravity in relation to standing is not well-established in the literature.Table 4). The findings of the current study lead us to conclude that 2-g centrifugation at feet via SAHC has potential to evoke autonomic control of blood pressure analogous to standing, therefore, a potential training tool toward reducing orthostatic intolerance in astronauts on their return to Earth.In the current article, we investigated the response of autonomic control of blood pressure during centrifugation (1-g and 2-g) in relation to standing. While no difference was observed in the autonomic control of blood pressure between standing and centrifugation, the blood pressure regulatory indices during standing and centrifugation (mostly 2-g) were significantly different from baseline (NG and AB conceived the centrifuge study and designed the experiment. AV and KT conceived the data analysis steps. MB, NG, and AB acquired the data. DX preprocessed the data. AV performed the data and statistical analyses, wrote the manuscript, and created the figures and tables. AV, DX, AG, NG, AB, and KT interpreted the results. All authors contributed to manuscript editing and approved the final version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate not only image-forming vision like other ganglion cells, but also non-image-forming physiological responses to light such as pupil constriction and circadian photoentrainment. All ipRGCs respond to light through their endogenous photopigment melanopsin as well as rod/cone-driven synaptic inputs. A major knowledge gap is how melanopsin, rods, and cones differentially drive ipRGC photoresponses and image-forming vision. We whole-cell-recorded from M4-type ipRGCs lacking melanopsin, rod input, or cone input to dissect the roles of each component in ipRGCs' responses to steady and temporally modulated (≥0.3 Hz) lights. We also used a behavioral assay to determine how the elimination of melanopsin, rod, or cone function impacts the optokinetic visual behavior of mice. Results showed that the initial, transient peak in an M4 cell's responses to 10-s light steps arises from rod and cone inputs. Both the sustainability and poststimulus persistence of these light-step responses depend only on rod and/or cone inputs, which is unexpected because these ipRGC photoresponse properties have often been attributed primarily to melanopsin. For temporally varying stimuli, the enhancement of response sustainedness involves melanopsin, whereas stimulus tracking is mediated by rod and cone inputs. Finally, the behavioral assay showed that while all three photoreceptive systems are nearly equally important for contrast sensitivity, only cones and rods contribute to spatial acuity. Opn4) and an invertebrate-like phototransduction cascade to generate excitatory responses to light  use the photopigment melanopsin  in which both copies of the melanopsin open reading frame are replaced by the gene encoding Cre recombinase  2 followed by cervical dislocation, and its eyes were enucleated and put in Ames' medium gassed with 95% O2 and 5% CO2. Under infrared-based night vision viewers, the retinas were isolated from the retinal pigment epithelium (RPE), the vitreous was removed using forceps, and each retina was cut into quadrants. A quadrant was flattened on the bottom of a superfusion chamber with the ganglion cell side up and superfused by bubbled Ames' medium at ~3 mL min−1, with temperature maintained at 33°C. The ganglion cell layer was viewed under infrared transillumination, and whole-cell recordings were made from the largest somas using an internal solution containing (in mM) 120 K-gluconate; 5 NaCl; 4 KCl; 10 HEPES; 2 EGTA; 4 Mg-ATP; 0.3 Na-GTP; 7 Tris-phosphocreatine; ~0.1% Lucifer Yellow for visualizing cellular morphologies; and KOH to set pH at 7.3.A mouse was dark-adapted overnight before the day of an experiment. Under dim red light, the mouse was euthanized using CO−2 s−1, with a mean intensity of 10.8 log photons cm−2 s−1 which is in the high mesopic range  and then by post-recording visualization of its morphology : 0.3, 0.4, 0.5, 0.7, 0.95, 1.3, 1.9, 2.45, 3.1, 4.1, 5.3, 6.3, 7.2, 8.9, 10.7, 13.9, 16.7, 19.6, 23.1, 27.5, and 31.7. This stimulus was identical to the “high contrast” sum-of-sinusoids stimulus described in , action potentials in the whole-cell recordings were first filtered out using a 20 Hz low-pass filter (for the light-step and flicker responses) or a 40 Hz low-pass filter (for the 21-frequency responses). Six parameters were analyzed:For analyzing graded membrane potentials , and cone-functionless (Gnat2cpfl3) M4 cells, and population averages of the recordings are shown in Figure −2 s−1 evoked a clear response in the rod-functionless cells, suggesting it is above the cone threshold. To estimate the melanopsin activation threshold, we recorded from wild-type, Gnat1−/− and Gnat2cpfl3 M4 cells in the presence of the rod/cone signaling blockers L-(+)-2-amino-4-phosphonobutyric acid (50 μM), 6,7-dinitroquinoxaline-2,3-dione (40 μM), and D-(-)-2-amino-5-phosphonopentanoic acid (25 μM) , the slight hyperpolarizing response at 11.5 log photons cm−2 s−1 was previously shown to be likely a photoelectric artifact , whereas abolishing rod or cone function significantly reduced the peak response amplitude at several light intensities .Statistical comparisons between the wild type's and the other genotypes' light-step responses are shown in Figure left), while disrupting rod or cone input significantly altered this ratio at one and two intensities, respectively .In the second analysis, we calculated final-to-peak amplitude ratios to quantify the sustainability of the light-step responses—the more sustained a response is, the higher its final-to-peak ratio would be. Eliminating melanopsin did not significantly alter the final-to-peak ratio for any of the responses Figure left, whVm relative to the baseline 10–50 s after light offset; we excluded the first 10 poststimulus seconds from this analysis to avoid the transient hyperpolarization seen in many responses. The wild-type cells had large persisting depolarizations at the four highest light intensities , but neither the melanopsin-knockout cells  nor the cone-functionless cells  had significantly reduced poststimulus depolarizations.In the third analysis, we quantified the poststimulus persistence of the light-step responses by averaging s Figure . Abolishs Figure middle, s Figure left nors Figure right haVm. Figure middle), not in those without melanopsin  or cone input .Contrary to our finding, a previous study reported a near-abolition of the poststimulus spiking increase in melanopsin-knockout M4 cells . Representative recordings are shown in Figure columns). By contrast, melanopsin-only responses recorded during rod/cone block did not show any tracking of the pulses , indicating that synaptic input is necessary for pulse tracking.The remaining whole-cell recording experiments assessed M4 cells' responses to temporally varying light. In the first experiment we presented 20-s flickers containing square light pulses that alternated with darkness, at 3 frequencies  and 2 intensities (11.5 log and 14.5 log photons cmtop). At the lower intensity, wild-type cells tracked all three flicker frequencies more or less equally well, and tracking ability was improved in rod-functionless cells for all three frequencies .Statistical comparisons between the wild type's and the other genotypes' flicker responses are shown in Figure s Figure bottom.columns). To quantify the sustainedness of the flicker responses, we computed ratios of the peak amplitude in the final-pulse response to the peak amplitude in the first-pulse response. The only significant change in flicker response sustainedness was seen when melanopsin was knocked out, making the cells' responses less sustained for the 0.5 Hz flicker at the higher intensity .The various genotypes' averaged flicker responses seemed to show different degrees of sustainability  was chosen because it is just high enough to stimulate melanopsin to some degree  while probably low enough to avoid excessive bleaching of rods and cones. Fast Fourier transform was used to calculate each cell's response amplitude at each of the 21 frequencies found in the stimulus. As shown in Figure The previous experiment tested only three frequencies and all pulses were delivered against a dark background, but the visual scene typically contains far more temporal frequencies and much of vision occurs in light-adapted conditions. Thus, in the next experiment we presented a sum-of-sinusoids stimulus with 21 frequencies summated around a 10.8 log photons cmy Figure  to rapidAs mentioned in the Introduction, ipRGCs mediate not only non-image-forming behavioral responses but also pattern vision, as previously demonstrated using an optokinetic tracking assay , so rods were likely less desensitized in our recordings. By contrast, because melanopsin has a much higher threshold than rhodopsin and is comparatively resistant to photobleaching  could reflect the bandpass properties of rod-cone coupling.For M4 cells lacking cone input, the flicker and sum-of-sinusoids experiments yielded seemingly contradictory results: responses to the flickers were not significantly affected at 0.5, 2, or 5 Hz, but responses to the sum-of-sinusoids increased at all frequencies between 0.5 and 2.45 Hz. This can be explained by cones influencing rods via gap-junction coupling  was statistically insignificant. But a direct comparison is impossible because optokinetic tracking is driven by multiple types of ganglion cells , 20 s (for the flickers), and 23 s (for the sum-of-sinusoids stimulus) to minimize irreversible bleaching of rod and cone photopigments, as the isolated retinas used in this study did not have access to the RPE's retinoid cycle.Finally, we examined only M4 cells and the findings may not be applicable to all the other ipRGC types. For instance, melanopsin could conceivably contribute more prominently to the light responses of M1–M3 ipRGCs, which express this photopigment at higher levels than M4 cells (Berson et al., We have found that, with one exception, melanopsin is largely dispensable for the light response of M4 ipRGCs under our testing conditions, even though our stimuli included intensities above the melanopsin activation threshold. Rod and cone inputs contribute to several aspects of M4 cells' photoresponse, with the rod input being solely responsible for the post-illumination persistence of the response. At the behavioral level, all three photoreceptor classes are important for contrast sensitivity, whereas visual acuity is established only by rods and cones. Since we examined just the M4 type, future studies will be needed to ascertain whether our single-cell results are applicable to the other types of ipRGCs.KW designed research. All authors performed research. KH, XZ, and KW analyzed data. KW wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer ES and handling Editor declared their shared affiliation.


Boys performed better in sit-ups in 1 minute and standing long jump tests and had higher waist circumference values. Girls performed better in sit-and-reach test. Overall, boys had higher physical fitness z-score values, compared to girls. Younger boys had better physical fitness perception , compared to older ones . In girls, correlation coefficient was the highest in the 3rd grade , followed by the 2ndgrade , the 1stgrade  and the 4th grade . Our study shows moderate sex and age specific correlation between self-reported and estimated physical fitness in a large sample of adolescents.The main purpose of the study was to explore sex and age specific correlations between self-reported and estimated physical fitness. In this cross-sectional study, we recruited 1 036 secondary-school students (55.3% girls). Self-reported physical fitness was assessed on a 10-point scale, with a higher score indicating better physical fitness. We measured waist circumference, sit-ups in 1 minute, standing long jump and sit-and-reach test and calculated  Physical fitness is considered one of the most important health markers  and a siComponents of physical fitness include: (1) cardiorespiratory, (2) musculoskeletal, 3) motor and 4) body composition fitness [ motor an body comAnother way to estimate physical fitness is through self-reported perception. It is a cost-and-time effective way to collect the data. Previous studies have tried to examine the correlation and agreement between self-reported and objectively estimated physical fitness in youth ,5 and thAlthough adolescents represent a population of apparently healthy individuals, the level of physical fitness established in adolescence often persists later in life , showingst to 4th). Each class had ≈25 students. All students were considered healthy and were not affected by diseases. The selection criteria were: (1) active participation in physical education classes and (2) absence of injuries. According to the Croatian Bureau of Statistics for the year 2017 [age±SD = 16.3±1.1 yrs; mheight±SD = 1.74±0.1 m; mweight±SD = 64.7±12.4 kg; mbody-mass index±SD = 21.3±3.0 kg/m2; 55.3% girls). After the selection of each school and class, we contacted physical education teachers to help us organize the study and obtain the approval of the principal. The measurement protocol for the study lasted from January to March 2019. For ≈25 students, it took us 30 minutes in each physical education class to collect the data. Before the study began, all students had got familiar with aims, hypotheses and benefits for participation in the study. All procedures performed in this study were in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the leading author. Also, all participants and their parents/guardians provided written informed consent for participation in the study.In this cross-sectional study, participants were secondary-school students. At the first stage, we randomly selected 11  out of 86 secondary-schools currently operating in the city of Zagreb. At the second stage, we randomly selected one class representing each grade within the school  to 10 (excellent fitness)  and it hWe used EUROFIT Battery Fitness Test to assess the level of physical fitness in adolescents. These tests are considered reliable and valid instruments to measure the level of physical fitness in children and adolescents . Waist cWaist circumference: Each participant stood still in a standing position. We used anthropometric tape placed horizontally midway between the lower rib margin and the iliac crest at the end of normal expiration .Standing long jump: Each subject performed distance jumps from a standing start. While performing the jumps, the subjects were asked to bend their knees with their arms in front of them, parallel to the ground, then to swing both arms, push off vigorously and jump forward as far as possible, trying to land with their feet together and stay upright. The best out of two attempts was taken as the final score (expressed in centimetres) .Sit-ups in 1 minute: Trunk strength was assessed as the maximum number of sit-ups achieved in one minute. Children were seated on the floor, backs straight, hands clasped behind their neck, knees bent at 90° with heels and feet flat on the mat. They then lay down on their backs, shoulders touching the mat, and returned to the sitting position with their elbows out in front to touch their knees, keeping the hands clasped behind their neck the whole time. The total amount of correctly performed sit-ups in 60 seconds was the score .Sit-and reach test: Sitting on the floor or a mat, legs straight under the angle of 90º, the person being tested reached forward with the arms (hands overlapping). The distance of reach was measured in centimetres using a measuring non-elastic tape attached on the floor .Height and weight were collected using portable stadiometer  with accuracy nearest to 0.5 cm and 0.2 kg in accordance to the International Society for the Advancement of Kinanthropometry .st grade = 15 year old, 2nd grade = 16 year old, 3rd grade = 17 year old and 4th grade = 18 year old). Differences between normally and not normally distributed variables were analyzed using univariate analysis of variance (ANOVA) and Kruskal-Wallis one-way analysis of variance (H-test). Previous studies have shown sex and age differences in perceived and estimated physical fitness [z-scores for each physical fitness test. Then, we summed all z-score values. Of note, we also checked for multicollinearity between physical fitness tests using the variance inflation factor (VIF). The VIF value was <2 indicating no multicollinearity between physical fitness tests. Significance was set up at p≤0.05, and it was two sided (2-sided). All the analysis were performed in Statistical Package for Social Sciences Software, ver. 22 .Data are presented as mean (SD) for normally distributed or median (25–75 interquartile range) for not normally distributed variables. All variables were grouped according to sex (boys vs.girls) and age . Compared to objectively estimated physical fitness, boys reported higher values of perceived physical fitness (p<0.001) compared with girls. Differences between objectively estimated physical fitness and self-reported physical fitness according to age were not observed (p>0.05).Basic descriptive statistics of the study participants are presented in st grade r = -0.10, p = 0.300; 2nd grade r = 0.07, p = 0.504; 3rd grade r = -0.11, p = 0.199 and 4th grade r = 0.02, p = 0.875). Other objectively estimated physical fitness tests were significantly correlated with self-reported physical fitness in all grades . In girls, self-reported physical fitness was also not significantly correlated with waist circumference . As in boys, other objectively estimated physical fitness tests were significantly correlated with self-reported physical fitness in all grades .In boys, self-reported physical fitness was not significantly correlated with waist circumference , followed by the 4th grade , the 3rd grade  and the 2nd grade . When boys were observed as a group, the correlation coefficient between self-reported and objectively estimated physical fitness was 0.50 (p<0.001). In girls, the highest correlation between self-reported and objectively estimated physical fitness was observed in the 3rd grade , followed by the 2nd grade  and the 1st grade . The lowest correlation between the aforementioned variables was observed in the 4th grade . The total correlation coefficient between self-reported and objectively estimated physical fitness in girls was 0.41 (p<0.001)Sex and age specific correlations of self-reported and estimated overall physical fitness are presented in The main purpose of the study was to explore sex and age specific correlations between self-reported and estimated physical fitness in a large sample of adolescents. Our study shows moderate correlation between the two variables in both boys and girls and according to age.et al. [et al. [z-scores of a submaximal bicycle ergometer test, ergojump tests, a 30-s sit-up test, hand-grip test and a sit-and-reach test. The same study showed a significant correlation between self-reported and objectively estimated physical fitness (rboth sexes = 0.54), concluding that at group level their study participants estimated the level of physical fitness moderately well [r = 0.36–0.64), muscle strength (r = 0.30–0.51) and flexibility (r = 0.31–0.36) [Our results are similar to one previous study exploring the same correlations in youth . The siget al.  found a et al. –9. All oet al. ,7,9 as a [et al.  used morely well . Strøjerely well on the ot31–0.36) .Physical fitness is a multidimensional construct with several components taken into account . AlthougOur study has several limitations. First, by using a cross-sectional design, we cannot conclude the causality of the correlation. Second, to assess objectively estimated physical fitness, we only used musculoskeletal and body composition components, taking cardiorespiratory fitness out of the equation. As mentioned above, most of the previous studies have used cardiorespiratory fitness as a proxy of overall physical fitness and shown moderate correlation with self-reported measure ,7,9,21.BOur study shows moderate correlation between self-reported and estimated physical fitness in a large sample of Croatian adolescents. If such measure is used in clinical settings or population-based studies conducted among adolescents, objectively estimated fitness accounts for 25% and 17% of the variance in boys and girls self reported fitness respectively.S1 Raw Data(XLSX)Click here for additional data file.


Fifty patients with diffuse large B cell lymphoma (DLBCL) were collected as a control group.Fifty patients with ALCL, including 32 anaplastic lymphoma kinase (ALK)-positive patients and 18 ALK-negative patients, were enrolled. The positive detection, maximal standardized uptake value (SUV18F-FDG-avid tumours with a mean SUVmax of 19.4 ± 12.6. Most (76%) ALCL patients presented with stage III-IV disease, and nodal and extranodal involvement occurred in 74.0 and 72.0% of the patients, respectively. ALCL and DLBCL showed many similarities in tumour stage, 18F-FDG uptake and tumour involvement (P > 0.05), although the preferred extranodal organs of involvement  were different (P < 0.05). Compared to ALK-negative lesions, a higher uptake of 18F-FDG was found in the ALK-positive lesions . ALK-positive ALCL was more likely to involve the lymph nodes than ALK-negative ALCL , while ALK-negative ALCL was more prone to involve the extranodal organs compared to ALK-positive ALCL .ALCL lesions were demonstrated to be 18F-FDG-avid lymphoma with many imaging manifestations similar to DLBCL on PET/CT. The present study also showed that ALK expression actually influenced tumour 18F-FDG uptake and lesion distribution. These findings may be useful to improve the understanding of the biological characteristics of ALCL.The present study demonstrated that ALCL is a systemic  The frequency of this disease varies geographically, with the highest incidence in parts of Asia, including China; however, it is particularly rare in European and America [ALK, the Anaplastic Lymphoma Kinase gene, located on chromosome 2p23. As an independent prognostic index, ALCL with positive expression of ALK is more responsive to therapy and associated with a better prognosis than ALK-negative ALCL [Anaplastic large cell lymphoma (ALCL) is a rare CD30 America –3. Since America . Two subive ALCL , 3, 5–7.18F-FDG positron emission tomography/computed tomography (PET/CT) is an important functional imaging technique that has become a standard imaging modality in staging, treatment response evaluation and prognostic prediction for FDG-avid lymphomas, according to the updated guidelines for imaging in lymphoma [18F-FDG PET/CT have rarely been described, and the role of 18F-FDG PET/CT in evaluating this disease has not been well established, possibly due to the low incidence rate of the disease [18F-FDG PET/CT images of 50 patients with ALCL were retrospectively reviewed to describe the ALCL imaging manifestation. Fifty patients with DLBCL, the most common subtype of lymphoma, were selected as the control group to compare the differences between the two diseases. disease . In the The institutional review board of Nanfang Hospital, Southern Medical University, approved this retrospective study. The institutional review board waived the requirement for written informed consent because of the retrospective nature of the study.18F-FDG PET/CT for staging from November 2005 to April 2018 were enrolled in the present study. If an ALCL patient received anti-tumour therapy, had uncontrolled diabetes, or had a history of other malignancies, he or she was excluded from the present study. Clinical data were collected for all of the patients, including age, sex, Ann Arbor clinical stage and the presence of B symptoms . 18F-FDG PET/CT images were retrospectively reviewed with a special focus on tumour 18F-FDG uptake and the distribution of nodal and extranodal involvement. To compare the difference in imaging manifestations of ALCL on 18F-FDG PET/CT to DLBCL, 50 consecutive patients with DLBCL who received 18F-FDG PET/CT for staging from July 2017 to September 2018 were collected as a control group.218F-FDG PET/CTFifty consecutive patients with histopathologically confirmed ALCL who received 18F-FDG PET/CT scan was performed using a Biograph mCTx scanner  in 47 patients and using a Discovery LS  in 3 additional patients. The patients were instructed to fast for at least 5 h and had blood glucose levels less than 11.1 mmol/L. Whole-body PET/CT was performed approximately 60 min after the intravenous injection of 222–446 MBq  18F-FDG. CT data were acquired using the following parameters: voltage , current (automatic milliampere-ampere), pitch (0.55), single-coil rotation time (1.0 s) and layer thickness (3 mm). 3D PET data were then acquired with a 2 min/bed position for the Biograph mCTx scanner, and 2D PET data were then acquired with a 4 min/bed position for the Discovery LS scanner according the established protocol in our centre [.r centre , 11.18F-FDG, i.e., higher than the radioactivity in the mediastinal blood pool specifically in the aortic arch, were considered positive. A region of interest (ROI) was placed over the lesions to measure each SUVmax. Based on the patient-specific lymphoma involvement, the lesion with the highest uptake of 18F-FDG was selected as the target lesion for SUVmax analysis. For the lesion-based/district-based analysis, the sites of lymph node involvement were partitioned into the neck, supraclavicular fossa, axilla, hilar of lung, mediastinum, retroperitoneal area, parailiac common vessels, internal and external parailiac vessels, inguinal region, and upper and lower limbs. Each site was scored as “1”. For those sites with bilateral sides, positive lymph nodes found on one side were scored as “1”, while those on both sides were scored as “2”. Single site, multisite and widespread involvement was used to classify the lymph node involvement into a single site, 2~5 sites and more than 5 sites of positive lymph nodes.PET data were reconstructed using the ordered subset maximum expectation iterative method (OSEM) with CT information for attenuation correction. PET and CT images were transferred to the Xeleris or Syngo MMWP workstations for image alignment fusion and analysis. The PET, CT and fused PET/CT data were independently reviewed by two experienced nuclear medical physicians who were blinded to the expression of ALK status and other clinical data. Any initial difference of opinion was resolved by consensus. Lesions with abnormally increased uptake of 18F-FDG was considered a positive lesion for lymphoma, while on the CT images, a solid nodule, or mass, or thickened tissue were considered positive lesion.The positive findings on PET and on the CT of PET/CT were comparatively analysed, especially for extranodal involvement. The CT scans were acquired under a non-enhanced contrast condition. On the PET images, a focal uptake of According to the WHO classification diagnostic standard, the diagnosis of ALCL is established on morphologic analysis and immunohistochemical examination. The tumour is characterized by the presence of large cells with horseshoe-shaped nuclei, prominent nucleoli, with or without a paranuclear HOF growing in a cohesive growth pattern, strong and homogeneous CD30 expression in the membrane and a Golgi pattern on immunohistochemical analysis. The expression of ALK in the ALCLs was measured by FISH analysis using a Vysis ALK gene rearrangement assay kit. FISH results were evaluated according to the number of positive cells. ALK-positive staining was defined as positive when 25 per 50 cells were positively stained. When the specimens had between 5 and 25 positive cells, the number of counted cells increased to 100 cells, and ALK-positivity was defined by more than 50 positive cells. For the Vysis ALK gene rearrangement assay, if there were ≥ 15 positive cells, ALK rearrangement was considered positive. If there were less than 15 positive cells, the result was considered negative.t tests were used to compare the SUVmax between two groups. The quantitative staging difference, nodal involvement, lymph node lesion distribution, and extranodal organ involvement data were expressed in frequencies or percentages. Pearson chi-square tests or Fisher’s exact tests were used for the data analysis. A P value less than 0.05 was defined as statistically significant.IBM SPSS 19.0 software was used for the data analysis. Measurement data that conformed to the normal distribution were expressed as x ± s. Independent two-sample Of the 50 patients with ALCL, 32 were positive for ALK and 18 were negative for ALK, with a male to female ratio of 2.6:1 and a median age of 28 years (range from 3 to 76 years old). According to the Ann Arbor staging system, 12 patients were classified as stage I-II (24%), and 38 patients were classified as stage III-IV (76%); 34 patients (68%) had B symptoms.18F-FDG-avid lymphoma lesions in at least one site in 49 out of 50 patients with a positive detection rate of 98.0%. A false-negative result was observed in a 3-year-old boy who had a large tumour  in the abdomen with a mild uptake of 18F-FDG . The mean SUVmax of the ALCL lesions was 19.4 ± 12.6. Nodal involvement was found in 37 (74.0%) ALCL patients, and extranodal involvement was found in 36 ALCL patients (72.0%). On the PET/CT images, 11 patients presented with solitary tumours, while the other 39 patients had multiple or widespread lesions. Of the 50 ALCL patients, 23 (46%) had both nodal and extranodal involvement, whereas 14 patients (28%) had nodal involvement alone and 13 patients (26%) had extranodal involvement alone. The most common nodal involved zone was the neck, followed by the mediastinum, retroperitoneal zone, mesentery and peri-iliac zone. The most commonly involved extranodal organ was bone (22/50), followed by the skin (8/50), spleen (6/50) and parotid gland (6/50). 18F-FDG PET was demonstrated to be superior to CT for detecting extranodal involvement, notably bone involvement. The positive detection rate of PET was 100% for bone involvement, which was significantly higher than the 59.1% positive detection rate of CT . PET also showed more sensitivity in the detection of cutaneous involvement than CT .PET/CT detected 18F-FDG PET/CT . Nodal involvement was found in 74.0% of ALCLs, which was similar to the 80.0% nodal involvement observed in the DLBCLs  . When nodal lesions were divided into 3 patterns: single site, multisite and widespread involvement, no significant difference in nodal involvement patterns was observed between ALCL and DLBCL  . There was no significant difference in the number of extranodal lesions between ALCL and DLBCL . However, the favoured sites of extranodal organ involvement in ALCL showed some differences from the favoured sites in DLBCL. In DLBCL patients, the gastrointestinal tract was the most commonly involved organ (11/50), followed by the nasopharynx (9/50), spleen (9/50), bone (8/50), and tonsils (7/50), while the most commonly involved organ of ALCL was bone (22/50), followed by the skin (8/50), spleen (6/50) and parotid gland (6/50)  . For both nodal and extranodal involvement, more DLBCL patients were found to have simultaneous nodal and extranodal involvement than ALCL patients    Table .Table 118F-FDG positive, while 31/32 of ALK-positive ALCLs were 18F-FDG positive. More intense uptake of 18F-FDG was found in the ALK-positive lesions compared to that of ALK-negative lesions . Regarding lesion distribution, ALK-positive ALCL was found to be more likely to involve the lymph nodes than ALK-negative ALCL; 84.3% (27/32) of ALK-positive ALCLs were observed to have positive lymph nodes compared to 55.5% (10/18) of ALK-negative ALCLs . The number of 18F-FDG-avid lymph nodes was 7.0 ± 4.7 for ALK-positive ALCL, which was significantly higher than that (3.4 ± 2.1) for ALK-negative ALCL (P = 0.003). Nodal involvement alone was observed in 12/32 ALK-positive ALCLs, which was significantly more than the 2/18 ALK-negative ALCLs  . Extranodal involvement alone was more commonly found in ALK-negative ALCL compared to ALK-positive ALCL   , which was consistent with the results reported by Lee DY, et al. [18F-FDG in the lesions implies that PET/CT is a perfect modality for staging and treatment response evaluation for ALCL. In the present study, it was confirmed that 18F-FDG PET was more sensitive for detecting extranodal involvement compared to CT, notably for detecting bone and cutaneous involvement (P < 0.05). A similar result was reported by Ram-Wolff et al.; 18F-FDG PET was more sensitive than CT in detecting cutaneous lesions [The present study revealed that ALCL had intense uptake of The present study demonstrated that most ALCLs (76%) presented with an advanced stage (stage III-IV) at the first visit to the doctor. ALCL commonly invaded the lymph nodes 74.0%), and the extranodal organs (72.0%). In nearly half of the patients (46.0%), simultaneous involvement of both lymph nodes and extranodal organs was found on PET/CT in the present study. Among the extranodal organs, bone was demonstrated to be the most commonly involved organ, which is inconsistent with previously published articles have shown that the skin is the most common extranodal involvement site .0%, and . In our 18F-FDG uptake on PET/CT  with similar distributions of nodal involvement. Therefore, 18F-FDG PET/CT may play a similar important role in ALCL and DLBCL. Despite the above similarities, some differences were observed between ALCL and DLBCL. In the present study, it was found that ALCL was more likely to involve the bone. In contrast, the most commonly involved extranodal organ in DLBCL was the gastrointestinal tract, which was similar to the data reported by Prasanna Ghimire [2 = 4.058, P = 0.044). To the best of our knowledge, there have been no prior studies comparing these similarities and differences between ALCL and DLBCL. Therefore, the findings in the present study may be useful to improve the understanding of the biological characteristics of this rare subtype of lymphoma.Although ALCL and DLBCL have different cell origins, they both have a good treatment response and prognosis, which is different from other T cell lymphoma subtypes , 22. We  Ghimire . In addi18F-FDG uptake than that of ALK-negative ALCL , which was consistent with the results reported by Lee DY [18F-FDG uptake in ALCL tumours may not be an indicator of treatment response and prognosis because ALK-positive ALCL was always reported to have a better prognosis than ALK-negative ALCL [18F-FDG uptake [Translocations involving the anaplastic lymphoma kinase (ALK) and nucleophosmin (NPM) were first identified in ALCLs , 25. Suby Lee DY . Similary Lee DY , 28 lungy Lee DY . Howeverive ALCL , 30. ExpG uptake . Their sG uptake .18F-FDG uptake, we also found that there were some differences in nodal and extranodal involvement between ALK-positive and ALK-negative patients. ALK-positive ALCL was observed to be more likely to involve the lymph nodes than ALK-negative ALCL  and had more sites of positive lymph nodes . In contrast, ALK-negative ALCL was found to be more prone to involve the extranodal organs compared to ALK-positive ALCL . Therefore, we suggested that the less favourable survival outcomes of ALK-negative ALCL [In the present study, in addition to differences in There are some limitations to the present study. First, the patient sample size was small, and further research is warranted to confirm our findings. Second, due to the shortcomings of retrospective studies, a prospective study may be more useful to discover the imaging characteristics of ALCL.18F-FDG-avid tumour. ALCL and DLBCL had some similarities, such as advanced stage in most patients, high 18F-FDG uptake and a similar distribution of nodal lesions. However, ALCL and DLBCL had some differences in extranodal involvement, especially in the bone and the gastrointestinal tract. The present study also found that ALCL with ALK-positive cells had a higher 18F-FDG uptake than ALK-negative ALCL. In addition, ALK-positive ALCL was more likely to involve the lymph nodes and had more sites of lymph node positivity. In contrast, ALK-negative ALCL was more prone to involve the extranodal organs. The findings in the present study may be useful to improve the understanding of the biological characteristics of this rare subtype of lymphoma.The present study demonstrated that ALCL is a systemic,